The present invention relates to a drug delivery system for delivering a peptide or protein drug into the brain, more specifically a conjugate between a biologically active (hereinafter "bioactive") peptide or protein and a carrier peptide capable of transporting the peptide or protein into the brain by receptor-mediated transcytosis, by means of a receptor on the cerebral capillary endothelial cell.
By means of pores and intracellular cavities present in the capillaries of tissues such as muscles, hydrophilic drugs in the blood quickly migrate into tissue intercellular fluid via intercellular routes. However, drugs in the blood do not easily migrate from capillaries to the brain, except those of molecules smaller than about 200 daltons in molecular weight which are not ionized at physiological pH or highly soluble in fat, because the cerebral capillaries have less pores and have endothelial cells bound strongly by connective tissue. This histologic filtering mechanism, known as the blood-brain barrier (BBB), represents a hindrance in chemotherapy for cerebral disease. For example, interleukin 2 (IL-2), a cytokine produced by T-cells and playing a key role in T-cell differentiation and proliferation, has recently been suggested to proliferate oligodendrocytes, astrocytes and precursor cells thereof in the central nervous system. However, IL-2 is incapable of passing the blood-brain barrier because of its high molecular weight of 15,400; its clinical application is therefore difficult. Also, nerve growth factor (NGF), as a nerve nutrition factor, plays a key role in the survival of cholinergic nerve cells in the central nervous system, and is expected to serve as a therapeutic agent for dementia diseases such as Alzheimer's disease. However, NGF is also difficult to apply clinically because it is incapable of passing the blood-brain barrier because of its high molecular weight of about 13,000.
Various methods of drug delivery to the brain have been developed so far, including direct injection and molecular modification. As a method of molecular modification, Boda et al. attempted to increase the lipophilicity of a drug to enhance its permeation via intracellular routes (Japanese Unexamined Patent Publication No. 277662/1988; EP0293071). However, this method is limited to drugs of certain molecular weight and is difficult to apply when the subject bioactive substance is a high molecular weight peptide, especially a protein.
As another approach, there have been attempts to use chimeric peptides based on endocytosis by endogenous peptide whose receptor is present on the cerebral capillary endothelial cell, such as insulin, insulin-like growth factor (IGF) and transferrin. For example, Pardridge et al. reported on chimeric peptides capable of passing the blood-brain barrier, that were prepared by binding insulin etc. with nerve drugs (Japanese Patent PCT Publication No. 500901/1989; WO88/00834). In view of the cerebral migration rate, however, carrier peptide so such as insulin, must be administered at large doses, involving the risk of severe side effects associated with blood sugar level reduction.
Meanwhile, much research has been undertaken into the relationship between the amino acid sequence and structure of insulin and its affinity for hormone receptors.
Insulin is a polypeptide hormone of 6000 daltons comprising two short polypeptide chains, called chains A and B, bound via disulfide bonds. Chain A is a polypeptide chain of 21 amino acids, having internal disulfide crosslinkage, while chain B is a polypeptide chain of 30 amino acids. Chains A and B bind together via two disulfide crosslinkages. Although human insulin produced by genetic engineering has recently been used, porcine insulin is generally used in diabetics. Porcine insulin differs from human insulin solely in that the carboxyl-terminal Tyr in chain B (Tyr.sup.B30) is replaced by Ala.sup.B30. Given the fact that, in four patients with insulinemia (Phe.sup.B25 .fwdarw.Leu.sup.B25, Chicago; Phe.sup.B24 .fwdarw.Ser.sup.B24, Los Angels; Val.sup.A3 .fwdarw.Leu.sup.A3, Wakayama and Tochigi), insulin lacks hormone receptor binding capability, it is suggested that B chain C-terminal amino acids and A chain N-terminal amino acids play a key role in insulin binding with hormone receptors. Also, the components of the C-termunus of chain B, especially 25-position Phe (Phe.sup.B25) of chain B, are associated with the bioactive potency of insulin. Nakagawa et al. obtained a synthetic insulin analogue known as (B25-30) pentapeptide deficient-[Phe.sup.B25 -.alpha.-carboxyamide]insulin, which acts as potently as natural insulin, by removing the C-terminal pentapeptide from the chain B and amidating the carboxyl group of the resulting Phe.sup.B25 [Journal of Biological Chemistry, Vol. 261, pp. 7332-7341 (1986)]. They also showed that insulin analogues resulting from replacement of Phe.sup.B25 with two or three other amino acid residues, i.e., (B26-30) pentapeptide deficient-[Tyr.sup.B25 -.alpha.-carboxyamide]insulin and His.sup.B25 analogues, are about 2.7 to 3.0 times as potent as insulin, and that analogues resulting from (B25-30) hexapeptide deficiency, (B24-30) heptapeptide deficiency or (B23-30) octapeptide deficiency possess almost no insulin activity. Also, Pullen et al. studied the crystallographic structure of insulin, pointing out that a key to expression of insulin bioactivity is molecular geometric stabilization of the oleophilic receptor binding site, including three residues in chain A (Gly.sup.1, Tyr.sup.1, Asn.sup.21) and five residues in chain B (Val.sup.12, Tyr.sup.16, Phe.sup.24, Phe.sup.25, Tyr.sup.26) [Nature, Vol. 259, pp. 369-373 (1976)].
These studies have provided a great deal of information on insulin analogues showing blood sugar reducing action and affinity for hormone receptors, but there is no report of receptors on cerebral capillaries.
In allowing a bioactive peptide or protein to pass the blood-brain barrier to transport it into the brain, it is desirable to transport the peptide or protein uniformly into the brain with minimum side effects. However, there is no well-established mode for a transport of bioactive peptide or protein meeting these requirements.